To replicate the associations of leukocyte telomere length (LTL) with variants at four loci and to investigate their associations with coronary heart disease (CHD) and type II diabetes (T2D), in order to examine possible causal effects of telomere maintenance machinery on disease aetiology.
Four SNPs at three loci BICD1 (rs2630578 GγC), 18q12.2 (rs2162440 GγT), and OBFC1 (rs10786775 CγG, rs11591710 AγC) were genotyped in four studies comprised of 2353 subjects out of which 1148 had CHD and 566 T2D. Three SNPs (rs12696304 CγG, rs10936601G>T and rs16847897 GγC) at the TERC locus were genotyped in these four studies, in addition to an offspring study of 765 healthy students. For all samples, LTL had been measured using a real-time PCR-based method.
Only one SNP was associated with a significant effect on LTL, with the minor allele G of OBFC1 rs10786775 SNP being associated with longer LTL (β=0.029, P=0.04). No SNPs were significantly associated with CHD or T2D. For OBFC1 the haplotype carrying both rare alleles (rs10786775G and rs11591710C, haplotype frequency 0.089) was associated with lower CHD prevalence (OR: 0.77; 95% CI: 0.61–0.97; P= 0.03). The TERC haplotype GTC (rs12696304G, rs10936601T and rs16847897C, haplotype frequency 0.210) was associated with lower risk for both CHD (OR: 0.86; 95% CI: 0.75-0.99; P=0.04) and T2D (OR: 0.74; 95% CI: 0.61–0.91; P= 0.004), with no effect on LTL. Only the last association remained after adjusting for multiple testing.
Of reported associations, only that between the OBFC1 rs10786775 SNP and LTL was confirmed, although our study has a limited power to detect modest effects. A 2-SNP OBFC1 haplotype was associated with higher risk of CHD, and a 3-SNP TERC haplotype was associated with both higher risk of CHD and T2D. Further work is required to confirm these results and explore the mechanisms of these effects.
Recent evidence suggests that dendritic cells may play an important role in atherosclerosis. Based primarily on previous in vitro studies, we hypothesized that granulocyte macrophage colony-stimulating factor (GM-CSF)-deficient mice would have decreased dendritic cells in lesions.
Methods and Results
To test this, we characterized gene targeted GM-CSF−/− mice crossed to hypercholesterolemic low-density lipoprotein receptor null mice. Our results provide conclusive evidence that GM-CSF is a major regulator of dendritic cell formation in vivo. Aortic lesion sections in GM-CSF−/− low-density lipoprotein receptor null animals showed a dramatic 60% decrease in the content of dendritic cells as judged by CD11c staining but no change in the overall content of monocyte-derived cells. The GM-CSF–deficient mice exhibited a significant 20% to 50% decrease in the size of aortic lesions, depending on the location of the lesions. Other prominent changes in GM-CSF−/− mice were decreased lesional T cell content, decreased autoantibodies to oxidized lipids, and striking disruptions of the elastin fibers adjacent to the lesion.
Given that GM-CSF is dramatically induced by oxidized lipids in endothelial cells, our data suggest that GM-CSF serves to regulate dendritic cell formation in lesions and that this, in turn, influences inflammation, plaque growth and possibly plaque stability.
atherosclerosis; dendritic cells; elastin; GM-CSF; macrophages
genetics; models, theoretical; pharmacogenetics; systems biology; atherosclerosis; heart failure; mapping
Common cardiovascular diseases, such as atherosclerosis and congestive heart failure, are exceptionally complex, involving a multitude of environmental and genetic factors that often show nonlinear interactions as well as being highly dependent on sex, age, and even the maternal environment. Although focused, reductionistic approaches have led to progress in elucidating the pathophysiology of cardiovascular diseases, such approaches are poorly powered to address complex interactions. Over the past decade, technological advances have made it possible to interrogate biological systems on a global level, raising hopes that, in combination with computational approaches, it may be possible to more fully address the complexities of cardiovascular diseases. In this Review, we provide an overview of such systems-based approaches to cardiovascular disease and discuss their translational implications.
Many common diseases have an important inflammatory component mediated in part by macrophages. Here we used a systems genetics strategy to examine the role of common genetic variation in macrophage responses to inflammatory stimuli.
We examined genome-wide transcript levels in macrophages from 92 strains of the Hybrid Mouse Diversity Panel. We exposed macrophages to control media, bacterial lipopolysaccharide, or oxidized phospholipids. We performed association mapping under each condition and identified several thousand expression quantitative trait loci (eQTL), gene-by-environment interactions and several eQTL “hotspots” that specifically control LPS responses. We validated an eQTL hotspot in chromosome 8 using siRNA knock-down of candidate genes and identified the gene 2310061C15Rik, as a novel regulator of inflammatory responses in macrophages.
We have created a public database where the data presented here can be used as a resource for understanding many common inflammatory traits which are modeled in the mouse, and for the dissection of regulatory relationships between genes.
There is increasing clinical evidence that phospholipid oxidation products (Ox-PL) play a role in atherosclerosis. This review focuses on the mechanisms by which Ox-PL interact with endothelial cells, monocyte/macrophages, platelets, smooth muscle cells and HDL to promote atherogenesis. In the last few years major progress has been made in identifying these mechanisms. It has been recognized that Ox-PL promote phenotypic changes in these cell types that have long term consequences for the vessel wall. Individual Ox-PL responsible for specific cellular effects has been identified. A model of the configuration of bioactive truncated Ox-PL within membranes has been developed that demonstrates that the oxidized fatty acid moiety protrudes into the aqueous phase, rendering it accessible for receptor recognition. Receptors and signaling pathways for individual Ox-PL species are now determined and receptor independent signaling pathways identified. The effects of Ox-PL are mediated both by gene regulation and transcription independent processes. It has now become apparent that Ox-PL affects multiple genes and pathways some of which are pro-atherogenic and some are protective. However, at concentrations that are likely present in the vessel wall in atherosclerotic lesions, the effects promote atherogenesis. There have also been new insights on enzymes that metabolize Ox-PL and the significance of these enzymes for atherosclerosis. With the knowledge we now have on the regulation and effects of Ox-PL in different vascular cell types, it should be possible to design experiments to test the role of specific Ox-PL on the development of atherosclerosis.
Ox-PAPC; atherosclerosis; inflammation; phospholipids
Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4- to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.
atherosclerosis; obesity; genetics
Rationale and Objective
In this Emerging Science Review, we discuss a systems genetics strategy, which we call Gene Module Association Study (GMAS), as a novel approach complementing Genome Wide Association Studies (GWAS), to understand complex diseases by focusing on how genes work together in groups rather than singly.
The first step is to characterize phenotypic differences among a genetically diverse population. The second step is to use gene expression microarray (or other high throughput) data from the population to construct gene co-expression networks. Co-expression analysis typically groups 20,000 genes into 20–30 modules containing 10’s to 100’s of genes, whose aggregate behavior can be represented by the module’s “eigengene.” The third step is to correlate expression patterns with phenotype, as in GWAS, only applied to eigengenes instead of SNPs.
Results and Conclusions
The goal of the GMAS approach is to identify groups of co-regulated genes that explain complex traits from a systems perspective. From an evolutionary standpoint, we hypothesize that variability in eigengene patterns reflects the “good enough solution” concept, that biological systems are sufficiently complex so that many possible combinations of the same elements (in this case eigengenes) can produce an equivalent output, i.e. a “good enough solution” to accomplish normal biological functions. However, when faced with environmental stresses, some “good enough solutions” adapt better than others, explaining individual variability to disease and drug susceptibility. If validated, GMAS may imply that common polygenic diseases are related as much to group interactions between normal genes, as to multiple gene mutations.
systems genetics; genetics of complex diseases; scale-free networks; hybrid mouse diversity panel; computational biology
The protein phosphatase 1-like gene (PPM1l) was identified as causal gene for obesity and metabolic abnormalities in mice. However, the underlying mechanisms were unknown. In this report, we find PPM1l encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (PP2Ce) and has specific activity to basal and ER stress induced auto-phosphorylation of Inositol-REquiring protein-1 (IRE1). PP2Ce inactivation resulted in elevated IRE1 phosphorylation and higher expression of XBP-1, CHOP, and BiP at basal. However, ER stress stimulated XBP-1 and BiP induction was blunted while CHOP induction was further enhanced in PP2Ce null cells. PP2Ce protein levels are significantly induced during adipogenesis in vitro and are necessary for normal adipocyte maturation. Finally, we provide evidence that common genetic variation of PPM11 gene is significantly associated with human lipid profile. Therefore, PPM1l mediated IRE1 regulation and downstream ER stress signaling is a plausible molecular basis for its role in metabolic regulation and disorder.
IRE1; PPM1l; Protein phosphatase; ER stress; Adipogenesis
To fine map previously identified quantitative trait loci (QTL) affecting atherosclerosis in mice using association analysis.
Methods and Results
We recently showed that high-resolution association analysis using common inbred strains of mice is feasible if corrected for population structure. To utilize this approach for atherosclerosis, which requires a sensitizing mutation, we bred human apoB100 transgenic mice with 22 different inbred strains to produce F1 heterozygotes. Mice carrying the dominant transgene were tested for association with high-density SNP maps. Here we focus on high-resolution mapping of the previously described Ath30 locus on Chr 1. As compared to the previous linkage analysis, association improved the resolution of the Ath30 locus by more than an order of magnitude. Using expression quantitative trait locus analysis, we identified one of the genes in the region, Des, as a strong candidate.
Our high-resolution mapping approach accurately identifies and fine maps known atherosclerosis QTL. These results suggest that high-resolution genome-wide association analysis for atherosclerosis is feasible in mice.
We investigated the association of glycemia and 43 genetic risk variants for hyperglycemia/type 2 diabetes with amino acid levels in the population-based Metabolic Syndrome in Men (METSIM) Study, including 9,369 nondiabetic or newly diagnosed type 2 diabetic Finnish men. Plasma levels of eight amino acids were measured with proton nuclear magnetic resonance spectroscopy. Increasing fasting and 2-h plasma glucose levels were associated with increasing levels of several amino acids and decreasing levels of histidine and glutamine. Alanine, leucine, isoleucine, tyrosine, and glutamine predicted incident type 2 diabetes in a 4.7-year follow-up of the METSIM Study, and their effects were largely mediated by insulin resistance (except for glutamine). We also found significant correlations between insulin sensitivity (Matsuda insulin sensitivity index) and mRNA expression of genes regulating amino acid degradation in 200 subcutaneous adipose tissue samples. Only 1 of 43 risk single nucleotide polymorphisms for type 2 diabetes or hyperglycemia, the glucose-increasing major C allele of rs780094 of GCKR, was significantly associated with decreased levels of alanine and isoleucine and elevated levels of glutamine. In conclusion, the levels of branched-chain, aromatic amino acids and alanine increased and the levels of glutamine and histidine decreased with increasing glycemia, reflecting, at least in part, insulin resistance. Only one single nucleotide polymorphism regulating hyperglycemia was significantly associated with amino acid levels.
The liver X receptor (LXR) signaling pathway is an important modulator of atherosclerosis, but the relative importance of the two LXRs in atheroprotection is incompletely understood. We show here that LXRα, the dominant LXR isotype expressed in liver, plays a particularly important role in whole-body sterol homeostasis. In the context of the ApoE−/− background, deletion of LXRα, but not LXRβ, led to prominent increases in atherosclerosis and peripheral cholesterol accumulation. However, combined loss of LXRα and LXRβ on the ApoE−/− background led to an even more severe cholesterol accumulation phenotype compared to LXRα−/−ApoE−/− mice, indicating that LXRβ does contribute to reverse cholesterol transport (RCT) but that this contribution is quantitatively less important than that of LXRα. Unexpectedly, macrophages did not appear to underlie the differential phenotype of LXRα−/−ApoE−/− and LXRβ−/−ApoE−/− mice, as in vitro assays revealed no difference in the efficiency of cholesterol efflux from isolated macrophages. By contrast, in vivo assays of RCT using exogenously labeled macrophages revealed a marked defect in fecal sterol efflux in LXRα−/−ApoE−/− mice. Mechanistically, this defect was linked to a specific requirement for LXRα−/− in the expression of hepatic LXR target genes involved in sterol transport and metabolism. These studies reveal a previously unrecognized requirement for hepatic LXRα for optimal reverse cholesterol transport in mice.
atherosclerosis; nuclear receptor; cholesterol metabolism; apoliporotein
Coronary artery disease; genome-wide association; mouse atherosclerosis; myocardial infarction; plasma lipoproteins; systems genetics
The Systems Genetics Resource (SGR) (http://systems.genetics.ucla.edu) is a new open-access web application and database that contains genotypes and clinical and intermediate phenotypes from both human and mouse studies. The mouse data include studies using crosses between specific inbred strains and studies using the Hybrid Mouse Diversity Panel. SGR is designed to assist researchers studying genes and pathways contributing to complex disease traits, including obesity, diabetes, atherosclerosis, heart failure, osteoporosis, and lipoprotein metabolism. Over the next few years, we hope to add data relevant to deafness, addiction, hepatic steatosis, toxin responses, and vascular injury. The intermediate phenotypes include expression array data for a variety of tissues and cultured cells, metabolite levels, and protein levels. Pre-computed tables of genetic loci controlling intermediate and clinical phenotypes, as well as phenotype correlations, are accessed via a user-friendly web interface. The web site includes detailed protocols for all of the studies. Data from published studies are freely available; unpublished studies have restricted access during their embargo period.
database; genomics; systems biology; data integration; web services; data analysis
Atherosclerosis is a chronic inflammatory disease initiated by monocyte recruitment and retention in the vessel wall. An important mediator of monocyte endothelial interaction is the chemokine IL-8. The oxidation products of phospholipids, including Ox-PAPC, accumulate in atherosclerotic lesions and strongly induce IL-8 in human aortic endothelial cells (HAECs). The goal of this study was to identify the proximal events leading to induction of IL-8 by Ox-PAPC in vascular endothelial cells.
Methods and Results
In a systems genetics analysis of HAECs isolated from 96 different human donors, we showed that HBEGF transcript levels are strongly correlated to IL-8 induction by Ox-PAPC. The silencing and overexpression of HBEGF in HAECs confirmed the role of HBEGF in regulating IL-8 expression. HBEGF has been shown to be stored in an inactive form and activation is dependent on processing by a dysintegrin and metalloproteinases (ADAM) to a form that can activate EGF receptor (EGFR). Ox-PAPC was shown to rapidly induce HBEGF processing and EGFR activation in HAECs. Using siRNA we identified three ADAMs that regulate IL-8 induction and directly demonstrated that Ox-PAPC increases ADAM activity in the cells by substrate cleavage assay. We provide evidence for one mechanism of Ox-PAPC activation of ADAM involving covalent binding of Ox-PAPC to cysteine on ADAM. Free thiol cysteine analogs showed inhibition of IL-8 induction by Ox-PAPC, and both a cysteine analog and a cell surface thiol blocker strongly inhibited ADAM activity induction by Ox-PAPC. Using microarray analyses, we determined that this ADAM pathway may regulate as much as 30% of genes induced by Ox-PAPC in HAECs.
This study is the first report demonstrating a role for the ADAM-HBEGF-EGFR axis in Ox-PAPC induction of IL-8 in HAECs. These studies highlight a role for specific ADAMs as initiators of Ox-PAPC action and provide evidence for a role of covalent interaction of Ox-PAPC in activation of ADAMs.
Oxidized phospholipids; atherosclerosis; endothelium; metaloproteinase; HBEGF; EGFR
In diabetes, glucose toxicity affects different organ systems, including pancreatic islets where it leads to β-cell apoptosis, but the mechanisms are not fully understood. Recently, we identified thioredoxin-interacting protein (TXNIP) as a proapoptotic β-cell factor that is induced by glucose, raising the possibility that TXNIP may play a role in β-cell glucose toxicity.
RESEARCH DESIGN AND METHODS
To assess the effects of glucose on TXNIP expression and apoptosis and define the role of TXNIP, we used INS-1 β-cells; primary mouse islets; obese, diabetic BTBR.ob mice; and a unique mouse model of TXNIP deficiency (HcB-19) that harbors a natural nonsense mutation in the TXNIP gene.
Incubation of INS-1 cells at 25 mmol/l glucose for 24 h led to an 18-fold increase in TXNIP protein, as assessed by immunoblotting. This was accompanied by increased apoptosis, as demonstrated by a 12-fold induction of cleaved caspase-3. Overexpression of TXNIP revealed that TXNIP induces the intrinsic mitochondrial pathway of apoptosis. Islets of diabetic BTBR.ob mice also demonstrated increased TXNIP and apoptosis as did isolated wild-type islets incubated at high glucose. In contrast, TXNIP-deficient HcB-19 islets were protected against glucose-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and caspase-3, indicating that TXNIP is a required causal link between glucose toxicity and β-cell death.
These findings shed new light onto the molecular mechanisms of β-cell glucose toxicity and apoptosis, demonstrate that TXNIP induction plays a critical role in this vicious cycle, and suggest that inhibition of TXNIP may represent a novel approach to reduce glucotoxic β-cell loss.
Background: Network construction and analysis algorithms provide scientists with the ability to sift through high-throughput biological outputs, such as transcription microarrays, for small groups of genes (modules) that are relevant for further research. Most of these algorithms ignore the important role of non-linear interactions in the data, and the ability for genes to operate in multiple functional groups at once, despite clear evidence for both of these phenomena in observed biological systems.
Results: We have created a novel co-expression network analysis algorithm that incorporates both of these principles by combining the information-theoretic association measure of the maximal information coefficient (MIC) with an Interaction Component Model. We evaluate the performance of this approach on two datasets collected from a large panel of mice, one from macrophages and the other from liver by comparing the two measures based on a measure of module entropy, Gene Ontology (GO) enrichment, and scale-free topology (SFT) fit. Our algorithm outperforms a widely used co-expression analysis method, weighted gene co-expression network analysis (WGCNA), in the macrophage data, while returning comparable results in the liver dataset when using these criteria. We demonstrate that the macrophage data has more non-linear interactions than the liver dataset, which may explain the increased performance of our method, termed Maximal Information Component Analysis (MICA) in that case.
Conclusions: In making our network algorithm more accurately reflect known biological principles, we are able to generate modules with improved relevance, particularly in networks with confounding factors such as gene by environment interactions.
gene expression; ICMg; scale-free topology; MINE; GxE interactions
We report a systems genetics analysis of high density lipoproteins (HDL) levels in an F2 intercross between inbred strains CAST/EiJ and C57BL/6J. We previously showed that there are dramatic differences in HDL metabolism in a cross between these strains, and we now report co-expression network analysis of HDL that integrates global expression data from liver and adipose with relevant metabolic traits. Using data from a total of 293 F2 intercross mice, we constructed weighted gene co-expression networks and identified modules (subnetworks) associated with HDL and clinical traits. These were examined for genes implicated in HDL levels based on large human genome-wide associations studies (GWAS) and examined with respect to conservation between tissue and sexes in a total of 9 data sets. We identify genes that are consistently ranked high by association with HDL across the 9 data sets. We focus in particular on two genes, Wfdc2 and Hdac3, that are located in close proximity to HDL QTL peaks where causal testing indicates that they may affect HDL. Our results provide a rich resource for studies of complex metabolic interactions involving HDL.
Rare variant accumulation studies can implicate genes in disease susceptibility when a significant burden is observed in patients versus controls. Such analyses might be particularly useful for candidate genes that are selected based on experiments other than genome-wide association studies (GWAS). We sought to determine whether rare variants in non-GWAS candidate genes identified from mouse models and human Mendelian syndromes of hypertriglyceridemia (HTG) accumulate in patients with polygenic adult-onset HTG.
Methods and Results
We resequenced protein coding regions of 3 genes with established roles (APOC2, GPIHBP1, LMF1) and 2 genes recently implicated (CREB3L3 and ZHX3) in TG metabolism. We identified 41 distinct heterozygous rare variants, including 29 singleton variants, in the combined sample; in total, we observed 47 rare variants in 413 HTG patients versus 16 in 324 controls (OR=2.3; P=0.0050). Post hoc assessment of genetic burden in individual genes using three different tests suggested that the genetic burden was most prominent in the established genes LMF1 and APOC2, and also in the recently identified CREB3L3 gene.
These extensive resequencing studies show a significant accumulation of rare genetic variants in non-GWAS candidate genes among patients with polygenic HTG, and indicate the importance of testing specific hypotheses in large-scale resequencing studies.
hyperlipoproteinemia; genetics; apolipoproteins; lipoproteins; cardiovascular diseases
Adipose differentiation is a complex process controlled by a network of
transcription factors and co-regulators. We compared the global gene expression
patterns of adipogenic and nonadipogenic clones of 3T3-F442A preadipocytes and
identified the transcriptional cofactor, vestigial-like 3 (Vgll3), as an
inhibitor of adipogenesis. Vgll3 expression is down-regulated during terminal
adipocyte differentiation in vitro and negatively correlates with weight and
total fat mass in vivo. Furthermore, enforced Vgll3 expression inhibits the
differentiation of preadipocytes in vitro, whereas shRNA-mediated knockdown of
Vgll3 expression promotes differentiation. Expression of Vgll3 promoted the
expression of genes associated with bone and chondrocyte formation, suggesting
that Vgll3 participates in the decision of mesenchymal cells to proceed down the
adipocyte, bone, or cartilage lineages. The elucidation of factors involved in
specification of the adipocyte phenotype may aid in the identification of new
strategies for the treatment of metabolic disease.
mesenchymal differentiation; PPAR; lipid metabolism; nuclear receptor
Mutations of the orphan transporter ABCC6 (ATP-binding cassette, subfamily C, member 6) cause the connective tissue disorder pseudoxanthoma elasticum. ABCC6 was thought to be located on the plasma membrane of liver and kidney cells.
Mouse systems genetics and bioinformatics suggested that ABCC6 deficiency affects mitochondrial gene expression. We therefore tested whether ABCC6 associates with mitochondria.
Methods and Results
We found ABCC6 in crude mitochondrial fractions and subsequently pinpointed its localization to the purified mitochondria-associated membrane fraction. Cell-surface biotinylation in hepatocytes confirmed that ABCC6 is intracellular. Abcc6-knockout mice demonstrated mitochondrial abnormalities and decreased respiration reserve capacity.
Our finding that ABCC6 localizes to the mitochondria-associated membrane has implications for its mechanism of action in normal and diseased states.
PXE; vascular calcification; ABCC6/MRP6; MAM; mitochondria; cardiovascular disease
The osteoblast-lineage consists of cells at various stages of maturation that are essential for skeletal development, growth, and maintenance. Over the past decade, many of the signaling cascades that regulate this lineage have been elucidated; however, little is known of the networks that coordinate, modulate, and transmit these signals. Here, we identify a gene network specific to the osteoblast-lineage through the reconstruction of a bone co-expression network using microarray profiles collected on 96 Hybrid Mouse Diversity Panel (HMDP) inbred strains. Of the 21 modules that comprised the bone network, module 9 (M9) contained genes that were highly correlated with prototypical osteoblast maker genes and were more highly expressed in osteoblasts relative to other bone cells. In addition, the M9 contained many of the key genes that define the osteoblast-lineage, which together suggested that it was specific to this lineage. To use the M9 to identify novel osteoblast genes and highlight its biological relevance, we knocked-down the expression of its two most connected “hub” genes, Maged1 and Pard6g. Their perturbation altered both osteoblast proliferation and differentiation. Furthermore, we demonstrated the mice deficient in Maged1 had decreased bone mineral density (BMD). It was also discovered that a local expression quantitative trait locus (eQTL) regulating the Wnt signaling antagonist Sfrp1 was a key driver of the M9. We also show that the M9 is associated with BMD in the HMDP and is enriched for genes implicated in the regulation of human BMD through genome-wide association studies. In conclusion, we have identified a physiologically relevant gene network and used it to discover novel genes and regulatory mechanisms involved in the function of osteoblast-lineage cells. Our results highlight the power of harnessing natural genetic variation to generate co-expression networks that can be used to gain insight into the function of specific cell-types.
The osteoblast-lineage consists of a range of cells from osteogenic precursors that mature into bone-forming osteoblasts to osteocytes that are entombed in bone. Each cell in the lineage serves a number of distinct and critical roles in the growth and maintenance of the skeleton, as well as many extra-skeletal functions. Over the last decade, many of the major regulatory pathways governing the differentiation and activity of these cells have been discovered. In contrast, little is known regarding the composition or function of gene networks within the lineage. The goal of this study was to increase our understanding of how genes are organized into networks in osteoblasts. Towards this goal, we used microarray gene expression profiles from bone to identify a group of genes that formed a network specific to the osteoblast-lineage. We used the knowledge of this network to identify novel genes that are important for regulating various aspects of osteoblast function. These data improve our understanding of the gene networks operative in cells of the osteoblast-lineage.
Ox-PAPC accumulates in atherosclerotic lesions, is pro-atherogenic, and influences the expression of over 1000 genes in endothelial cells.
To elucidate the major pathways involved in Ox-PAPC action, we conducted a systems analysis of endothelial cell gene expression after exposure to Ox-PAPC.
Methods and Results
We used the variable responses of primary endothelial cells from 149 individuals exposed to Ox-PAPC to construct a network consisting of 11 groups of genes or modules. Modules were enriched for a broad range of GO pathways, some of which have not been previously identified as major Ox-PAPC targets. Further validating our method of network construction, modules were consistent with relationships established by cell biology studies of Ox-PAPC effects on endothelial cells. This network provides novel hypotheses about molecular interactions, as well as candidate molecular regulators of inflammation and atherosclerosis. We validate several hypotheses based on network connections and genomic association. Our network analysis predicted that the hub gene CHAC1 was regulated by the ATF4 arm of the unfolded protein response pathway and here we showed that ATF4 directly activates an element in the CHAC1 promoter. We show that variation in basal levels of HMOX1 contribute to the response to Ox-PAPC, consistent with its position as a hub in our network. We also identified GPR39 as a regulator of HMOX1 levels and showed that it modulates the promoter activity of HMOX1. We further showed that OKL38/OSGN1, the hub gene in the blue module, is a key regulator of both inflammatory and anti-inflammatory molecules.
Our systems genetics approach has provided a broad view of the pathways involved in the response of endothelial cells to Ox-PAPC and also identified novel regulatory mechanisms.
Endothelial Cells; Oxidized Phospholipids; Gene Expression; Heme Oxygenase; Network; Systems Genetics; Genome-Wide Association Studies